CN112970830A - 一种茶藨子提取物及其防腐用途 - Google Patents
一种茶藨子提取物及其防腐用途 Download PDFInfo
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- A23B4/20—Organic compounds; Microorganisms; Enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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Abstract
本发明提供了一种茶藨子提取物,它由包括如下内容的方法制备得到:取茶藨子乙酸乙酯提取物,上苯乙烯型弱极性大孔吸附树脂,以乙醇含量0‑20%的水洗脱,收集洗脱液,制备得到茶藨子提取物。狭果茶藨子提取物可以有效抑制蜡样芽孢杆菌生长繁殖,具有成为天然食品保鲜剂的潜力。
Description
技术领域
本发明属于天然植物提取物领域。
背景技术
随着食品科学领域的发展,食品安全逐渐成为食品工业中不可忽视的全球性问题,由微生物引起的食品腐败与疾病给世界各国造成了损失,目前广泛应用的化学防腐剂价格低廉,虽然可以抑制细菌生长,但对人体有一定的致畸、致癌和致突变作用。
蜡样芽孢杆菌(Bacillus cereus)是一种内生芽孢的革兰氏阳性杆状菌,寄生于土壤中,污染多种食品,如大米、蔬菜、鸡蛋、肉制品、乳制品等,蜡样芽孢杆菌是与食物中毒相关的食源性致病菌之一,能产生多种毒素导致腹泻和呕吐等症状,因此寻找高效安全的天然保鲜剂来应对食品腐败菌具有重大意义。
狭果茶藨子(Ribes stenocarpum Maxim)属虎耳草科植物,全世界约有160种,青海省有11种,1个变种。茶藨子为落叶灌木,生于山坡灌丛、云杉林和杂木林下或山沟中。研究发现,茶藨子的化学成分主要为:不饱和脂肪酸、有机酸、多糖、黄酮类化合物等。前期研究发现,青藏高原狭果茶藨子提取物对金黄色葡萄球菌、大肠杆菌等具有一定的抑制作用,具有成为新型多功能食品添加剂的潜力。但目前未见狭果茶藨子在对蜡样芽孢杆菌方面的活性。
发明内容
本发明提供了一种抗菌提取物,具体是提供了一种茶藨子提取物,它由包括如下内容的方法制备得到:
取茶藨子乙酸乙酯提取物,上苯乙烯型弱极性大孔吸附树脂,以乙醇含量0-20%的水洗脱,收集洗脱液,制备得到茶藨子提取物。
其中,所述茶藨子选自狭果茶藨子。
其中,所述苯乙烯型弱极性大孔吸附树脂选自AB-8型大孔吸附树脂。
其中,所述乙醇含量0-20%的水中,乙醇含量选自0、10%或20%。
其中,所述洗脱,选自单次洗脱或梯度洗脱。
所述单次洗脱,是指只采用一种溶剂进行洗脱,并收集该单一溶剂的洗脱液。例如,单独使用纯水(即乙醇含量为0的水)洗脱,收集水洗脱液;或者,单独使用乙醇含量20%的水洗脱,收集该洗脱液;等等。
本发明所述梯度洗脱,是指依次使用乙醇含量从低到高的水进行多次洗脱,收集其中一种或多种溶剂的洗脱液。例如,可以依次使用乙醇含量0、10%、20%的水进行洗脱,收集三种不同溶剂的洗脱液,或者仅收集其中之一的洗脱液;或者,可以依次使用乙醇含量0、20%的水进行洗脱,收集两种不同溶剂的洗脱液,或者仅收集其中之一的洗脱液;等等。
本发明还提供了茶藨子提取物的制备方法,它包括如下内容:
取茶藨子乙酸乙酯提取物,上苯乙烯型弱极性大孔吸附树脂,以乙醇含量0-20%的水洗脱,收集洗脱液,制备得到茶藨子提取物。
其中,所述茶藨子选自狭果茶藨子。
其中,所述苯乙烯型弱极性大孔吸附树脂选自AB-8型大孔吸附树脂。
其中,所述乙醇含量0-20%的水中,乙醇含量选自0、10%或20%。
其中,所述洗脱,选自单次洗脱或梯度洗脱。
本发明还提供了上述茶藨子提取物在制备抗细菌产品中的用途。
其中,所述细菌选自蜡样芽孢杆菌。
本发明还提供了一种抗菌产品,它包括如上所述的茶藨子提取物。
本发明还提供了一种防腐剂,它包括如上所述茶藨子提取物。本发明研究发现,狭果茶藨子提取物DW1能抑制生猪肉中腐败菌的生长,延缓其腐败变质,保护肉色稳定,延长货架期,保鲜效果良好。
本发明中所述茶藨子乙酸乙酯提取物,可以直接购买已有的茶藨子乙酸乙酯提取物,也可以通过常规方法进行提取,例如,以乙酸乙酯直接提取茶藨子;或者,以乙醇、含水乙醇、甲醇、含水甲醇提取茶藨子,再使用乙酸乙酯进行萃取,或者使用系统溶剂法,依次用极性从低到高的有机溶剂进行萃取,取乙酸乙酯萃取层即可。
如前所述“含水乙醇”或“含水甲醇”,乙醇或甲醇含量在80%以上,例如,80%、85%、90%、95%等等。
本发明中所述乙醇含量、甲醇含量,是指体积分数。
本发明中所述提取,可以采取本领域常规的方法,例如回流、浸渍、超声、微波辅助等等。
本发明在制备产品时,除了活性成分外,还可以加入辅料或/和辅助性成分。
本发明中所述的“辅料”,是药物制剂中除主药以外的一切附加材料的总称,辅料应当具备如下性质:(1)对人体无毒害作用,几无副作用;(2)化学性质稳定,不易受温度、pH、保存时间等的影响;(3)与主药无配伍禁忌,不影响主药的疗效和质量检查;(4)不与包装材料相互发生作用。
所述“辅助性成分”,它具有一定生理活性,但该成分的加入不会改变上述组合物在疾病治疗过程中的主导地位,而仅仅发挥辅助功效,这些辅助功效仅仅是对该成分已知活性的利用,是医药领域惯用的辅助治疗方式。若将上述辅助性成分与本发明组合物配合使用,仍然应属于本发明保护的范围。
药学上可以接受的辅料,包括但不限于纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明中,以青藏高原狭果茶藨子为原料,探究其对蜡样芽孢杆菌的抑菌活性及作用机制,利用纸片扩散法和二倍稀释法评价抑菌活性,通过测定膜蛋白荧光光谱、碱性磷酸酶、Na+/K+-ATP酶、呼吸链脱氢酶活力等探究抑菌机制。结果表明:狭果茶藨子提取物对蜡样芽孢杆菌有明显的抑制作用,狭果茶藨子提取物可改变蜡样芽孢杆菌细胞膜膜蛋白的构象,造成核酸和蛋白质泄露,破坏细胞壁,通过抑制苹果酸脱氢酶、呼吸链脱氢酶的活力抑制呼吸作用和能量代谢,同时提高Na+/K+-ATP酶、Ca2+/Mg2+-ATP酶和T-ATP1酶活力,激活了膜结合离子通道,改变细胞膜内外离子浓度以抵抗不利环境的影响;扫描电镜结果表明提取物导致蜡样芽孢杆菌细胞严重变形,表面有异常凸起和凹陷,说明狭果茶藨子可以有效抑制蜡样芽孢杆菌生长繁殖,且效果与已知的化学抗菌剂或抗生素相当、甚至更好,具有成为天然食品保鲜剂的潜力。
附图说明
图1不同提取物浓度对蜡样芽孢杆菌抑菌圈直径大小
图2不同浓度DW1对蜡样芽孢杆菌抑菌圈直径大小,其中,1:100mg/mL;2:200mg/mL;3:300mg/mL;a:氨苄西林;b:青霉素;c:300mg/mL脱氢乙酸钠;d:300mg/mL苯甲酸钠)
图3不同浓度狭果茶藨子提取物下蜡样芽孢杆菌生长曲线
图4不同浓度KI下蜡样芽孢杆菌膜蛋白氨基酸残基荧光光谱
图5不同浓度狭果茶藨子提取物下蜡样芽孢杆菌膜蛋白氨基酸残基荧光光谱
图6不同浓度狭果茶藨子提取物下蜡样芽孢杆菌AKP活力影响
图7不同浓度狭果茶藨子提取物下蜡样芽孢杆菌核酸及蛋白质泄露
图8不同浓度狭果茶藨子提取物对蜡样芽孢杆菌ATP活力的影响
图9不同浓度狭果茶藨子提取物对蜡样芽孢杆菌苹果酸脱氢酶活力的影响
图10不同浓度狭果茶藨子提取物对蜡样芽孢杆菌呼吸链脱氢酶活力影响
图11不同浓度狭果茶藨子提取物对蜡样芽孢杆菌微观形态影响
图12狭果茶藨子提取物DW1对生猪肉菌落总数的影响
图13狭果茶藨子提取物DW1对生猪肉高铁肌红蛋白比例的影响
具体实施方式
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
实施例1本发明提取物的制备
取狭果茶藨子乙酸乙酯提取物,上AB-8大孔树脂,用水进行洗脱,收集洗脱液,除去或不除去溶剂,即得本发明提取物。
实施例2本发明提取物的制备
取狭果茶藨子乙酸乙酯提取物,上AB-8大孔树脂,用20%乙醇进行洗脱,收集洗脱液,除去或不除去溶剂,即得本发明提取物。
实施例3本发明提取物的制备
取狭果茶藨子乙酸乙酯提取物,上AB-8大孔树脂,依次用水、20%乙醇进行洗脱,收集20%乙醇洗脱液,除去或不除去溶剂,即得本发明提取物。
实施例4本发明提取物的制备
取狭果茶藨子乙酸乙酯提取物,上AB-8大孔树脂,依次用水、20%乙醇进行洗脱,收集水和20%乙醇洗脱液,除去或不除去溶剂,即得本发明提取物。
实施例5本发明抗菌产品的制备
取本发明提取物,加入适量的辅料,制备抗菌产品。
实施例6本发明提取物的抗菌活性实验
1材料和方法
1.1材料与试剂
狭果茶藨子产自青海互助珠固乡,蜡样芽孢杆菌(CMCC(B)63303)购自上海鲁微科技有限公司。超微量ATP、苹果酸脱氢酶、琥珀酸脱氢酶、碱性磷酸酶试剂盒购自南京建成生物科技有限公司。碘硝基氯化四氮唑蓝(INT),上海源叶生物科技有限公司。
1.2仪器与设备
荧光分光光度计(RF-6000),日本岛津公司;微生物生长曲线仪(Micro-GCM),德国BMG公司;扫描电镜(JSM-6610LV),日本电子公司;显微细胞分析、菌落计数抑菌圈测量联用仪(MF1),杭州迅数科技有限公司。
1.3方法
1.3.1狭果茶藨子粗提物的制备
狭果茶藨子低温烘干,粉碎,过60目筛,取1000g样品加入95%乙醇回流提取2h,提取两次,过滤后合并滤液并浓缩,得到总粗提物80g,分别用乙酸乙酯、正丁醇萃取,制备乙酸乙酯提取物、正丁醇提取物、水提取物。
采用AB-8大孔树脂进一步分离纯化乙酸乙酯提取物(40g),分别用蒸馏水、20%、40%、60%、80%、100%乙醇梯度洗脱,收集不同极性洗脱液,浓缩,低温冷冻干燥,得组分DW1、DW2、DW3、DW4、DW5、DW6,其中DW1为19g,4℃密封储存备用。
1.3.2抑菌活性测定
将蜡样芽孢杆菌活化后,用无菌生理盐水稀释,比浊至0.5麦氏单位,配制不同浓度狭果茶藨子提取物,并放入药敏纸片浸泡12h,采用纸片扩散法进行抑菌实验,菌落计数器测量抑菌圈直径。
1.3.3最低抑菌浓度和最低杀死浓度测定
参照KANG等的方法测定最低抑菌浓度和最低杀死浓度。Kang Shimo,Kang FanhuaShi Xinyang,et al.Antibacterial activity and mechanism of lactobionic acidagainst Pseudomonas fluorescens and Methicillin-resistant Staphylococcusaureus and its application on whole milk[J].Food Control,2020,108:106876.
1.3.4生长曲线测定
将细菌接至含有提取物的96孔板,使终浓度分别为1/16MIC、1/8MIC、1/4MIC、1/2MIC、MIC、MBC,置于微生物生长曲线检测系统中培养35h(37℃200r/min),每30min测定一次,绘制生长曲线。
1.3.5细胞膜膜蛋白测定
参照WANG的方法测定蜡样芽孢杆菌细胞膜蛋白的荧光光谱。WANG Langhong,WANGMansheng,ZENG X,et al.Membrane and genomic dna dual-targeting of citrusflavonoid naringenin against staphylococcus aureus[J].Integr Biol(Camb),2017,9(10):820-829.
1.3.6碱性磷酸酶测定
参照SONG的方法测定碱性磷酸酶活力。Mingzhu Song,Xirui Wang,Canquan Mao,et al.The discovery of a potential antimicrobial agent:the novel compoundnatural medicinal plant fermentation extracts against candida albicans[J].IOPConference Series:Materials Science and Engineering,2018,301(1):012026.
1.3.7核酸及蛋白质泄漏试验
参照LIU的方法测定核酸蛋白质泄露。LIU xue,CAI Jiaxin,CHEN Haiming,etal.Antibacterial activity and mechanism of linalool against pseudomonasaeruginosa[J].Microb Pathog,2020,141:103980.
1.3.8ATP酶测定
参照WANG的方法测定蜡样芽孢杆菌中Na+/K+-ATP酶、Ca2+/Mg2+-ATP酶、T-ATP酶的活力。Wang Baoying,Gao Xiaolin,Liu Baoguang,et al..Protective effects ofcurcumin against chronic alcohol-induced liver injury in mice throughmodulating mitochondrial dysfunction and inhibiting endoplasmic reticulumstress[J].Food&nutrition research,2019,63:3597.
1.3.9呼吸链脱氢酶和苹果酸脱氢酶活力测定
取生长期的蜡样芽孢杆菌与1mL提取物混合,37℃120r/min条件下震荡培养1h,菌悬液离心并用生理盐水清洗,加入2.7mL PBS和0.3mL INT混合后于室温下避光暗处理1h,490nm下测定吸光度值。
将蜡样芽孢杆菌接种到含不同浓度提取物的MH肉汤中,37℃120r/min培养6h,离心收集细菌,PBS洗涤3次,组织匀浆机匀浆,使用苹果酸脱氢酶检测试剂盒检测。
1.3.10扫描电镜观察
参照Li等的方法观察细胞微观变化。LI Yingqiu,HAN qing,FENG Jianling,etal.Antibacterial characteristics and mechanisms ofε-poly-lysine againstescherichia coli and staphylococcus aureus[J].Food Control,2014,43:22-27.
1.4数据分析
ˉ
采用Excel 2010进行数据整理及作图,实验统计数据以平均值±标准差(x±SD,n=3)表示。使用SPSS 21.0进行数据处理。
2结果与分析
2.1抑菌活性筛选
狭果茶藨子提取物对蜡样芽孢杆菌抑菌效果如图1所示,抑菌圈直径大小关系为:乙酸乙酯提取物>正丁醇提取物>水提取物,抑菌活性与提取物浓度成正比,当乙酸乙酯提取物浓度为400mg/mL时,抑菌圈直径19.21±0.11mm,表现为高敏,效果明显优于正丁醇提取物和水提取物,说明乙酸乙酯提取物为抑制蜡样芽孢杆菌的有效活性部位。
乙酸乙酯提取物经AB-8大孔吸附树脂分离纯化后,得到6个组分,其抑菌效果如表1所示,其中DW1组分抑菌活性最强,其次为DW2组分,其他组分则无抑菌作用;当DW1浓度为300mg/mL时,抑菌圈直径18.54±0.52mm,表现为高敏,与食品中常用的防腐保鲜剂丙酸钠、山梨酸钾、苯甲酸钠和脱氢乙酸钠比较,丙酸钠与山梨酸钾对蜡样芽孢杆菌无抑制作用,苯甲酸钠、脱氢乙酸钠、青霉素(PEN)和氨苄西林(AMP)抑菌圈直径均小于DW1,抑菌圈大小如图2所示,因此后期抑菌机制试验选用DW1组分进行,抑菌效果DW1>脱氢乙酸钠>苯甲酸钠>山梨酸钾=丙酸钠,DW1抑菌效果优于阳性对照药氨苄西林(10/10μg)和青霉素(10U)。
表1不同组分对蜡样芽孢杆菌抑菌圈直径大小
(注:抑菌圈大于20mm为极敏;15-20mm为高敏;10-15mm为中敏;7-9mm为低敏。)
2.2最低抑菌浓度和最低杀菌浓度
狭果茶藨子提取物对蜡样芽孢杆菌具有较强抑制作用,实验结果表明,最低抑菌浓度(MIC)为3.13mg/mL,最低杀菌浓度(MBC)为6.25mg/mL,为MIC的两倍,空白对照无抑菌作用。
2.3蜡样芽孢杆菌生长曲线测定结果
狭果茶藨子提取物对蜡样芽孢杆菌生长影响如图3所示,对照组迟缓期为1h,随后进入对数期,5h后进入稳定期,35h内未进入衰亡期,MBC和MIC组吸光值变化较小,即细菌生长较少,1/2MIC组第16h进入生长期,持续至26h,1/4MIC组2h时进入生长期,12h进入稳定期,1/8MIC组与1/16MIC与对照相差较小,抑制作用不明显,结果表明,当浓度为MIC和MBC时,提取物抑制蜡样芽孢杆菌生长繁殖,当浓度为1/2MIC和1/4MIC时,表现为迟缓期和对数生长期延长。
2.4对蜡样芽孢杆菌膜蛋白的影响
膜蛋白是细胞的重要组成部分,在物质运输、细胞识别、信号传导等方面具有重要功能。膜蛋白中含有苯丙氨酸(Phe)、酪氨酸(Tyr)和色氨酸(Trp)等氨基酸,可以在膜蛋白中发出荧光。碘化钾(KI)是一种荧光淬灭剂,可以定性确定Phe、Trp、Tyr残基在蛋白质中的位置。图4显示了不同浓度KI处理蜡样芽孢杆菌膜蛋白氨基酸残基的荧光光谱,KI浓度从上到下依次为0.005、0.01、0.015、0.02、0.025、0.03mol/L。结果表明,随着KI浓度增加苯丙氨酸荧光淬灭效果明显,色氨酸和酪氨酸荧光淬灭效果不明显,表明色氨酸和酪氨酸残基主要位于细胞膜内部。图5显示了不同浓度狭果茶藨子提取物对蜡样芽孢杆菌膜蛋白荧光光谱的影响,提取物浓度从上到下依次为1/32MIC、1/16MIC、1/8MIC、1/4MIC、1/2MIC、MIC、MBC,结果表明,随着提取物浓度增加各氨基酸荧光强度均逐渐下降、当浓度为MIC和MBC时,伴随明显红移,这表明狭果茶藨子提取物可能与膜蛋白结合,改变了蜡样芽孢杆菌细胞膜膜蛋白的结构和构象,使Phe、Trp和Tyr残基变得更加亲水,导致细胞膜功能障碍和细胞损伤。
2.5碱性磷酸酶活力测定结果
碱性磷酸酶(AKP)存在于细胞壁和细胞膜之间,当细胞壁被破坏,碱性磷酸酶会流出。不同浓度狭果茶藨子提取物对蜡样芽孢杆菌碱性磷酸酶活力影响如图6所示,对照组AKP活力为1.11金氏单位/mgprot,MBC组AKP活力为3.60金氏单位/mgprot,是对照组的3.24倍,随着提取物浓度的增大,AKP活力增加,说明蜡样芽孢杆菌细胞壁受损越严重,碱性磷酸酶流出越多。
2.6核酸及蛋白质泄露结果
核酸和蛋白质对于细胞生长、新陈代谢、维持细胞功能有重要作用,细胞膜损伤,胞内物质就会泄露,细胞膜的完整性对细菌发育至关重要。狭果茶藨子提取物对蜡样芽孢杆菌核酸及蛋白质泄露影响结果如图7所示,由图可知,对照组核酸及蛋白质几乎不发生泄露,12h内OD280值、OD260值变化不大,经狭果茶藨子提取物处理后,蜡样芽孢杆菌核酸蛋白质泄露与样品浓度呈正相关,其中MBC组OD280值从0.01增加至0.16,OD260值从0.01增加至0.15,说明随着时间和浓度增加,蜡样芽孢杆菌核酸及蛋白质泄露程度加剧,即细胞膜破损越严重,进而影响细胞生长和新陈代谢,最终导致蜡样芽孢杆菌死亡。
2.7ATP酶活力测定结果
Na+/K+-ATP酶即钠钾泵,能促进ATP的水解,能够调节细胞内钠/钾离子浓度,为Na+、K+的跨膜运输提供能量,从而保持细胞的静息电位;Ca2+/Mg2+-ATP酶主要功能是建立跨膜的离子梯度、维持细胞内外离子平衡及渗透压平衡。狭果茶藨子提取物对蜡样芽孢杆菌ATP酶的影响结果如图8所示,随着提取物浓度升高,三种酶活力均升高,对照组Na+/K+-ATP酶活力为11.99U/mgprot,Ca2+/Mg2+-ATP酶活力为3.79U/mgprot,T-ATP酶活力为15.65U/mgprot,MBC组Na+/K+-ATP酶活力27.69U/mgprot,为对照组的2.3倍,Ca2+/Mg2+-ATP酶活力8.34U/mgprot,为对照组的2.2倍,T-ATP酶活力59.47U/mgprot,为对照组的3.8倍,以上数据表明,狭果茶藨子提取物能有效提高Na+/K+-ATP酶、Ca2+/Mg2+-ATP酶及T-ATP酶的活力,激活蜡样芽孢杆菌膜结合离子通道,显著破坏细胞膜,增加细胞膜的通透性;细菌通过改变细胞膜内外离子浓度以抵抗不利环境的影响,保持细胞内外电化学梯度的平衡。
2.8苹果酸脱氢酶活力测定结果
苹果酸脱氢酶是生物糖代谢的关键酶之一,参与能量代谢以及活性氧代谢等多种生理活动。狭果茶藨子提取物对蜡样芽孢杆菌苹果酸脱氢酶活力影响如图9所示,随着提取物浓度增加酶活力降低,对照组酶活力24.86U/mgprot,MBC组酶活力2.41U/mgprot,仅为对照组的十分之一,说明狭果茶藨子提取物可能通过抑制蜡样芽孢杆菌苹果酸脱氢酶活力,破坏细胞能量代谢,导致菌体细胞无法进行正常生理活动。
2.9呼吸链脱氢酶活力测定结果
呼吸链脱氢酶是TCA循环中的关键酶,反映了细胞呼吸作用的强度和生成ATP的能力。狭果茶藨子提取物对蜡样芽孢杆菌呼吸链脱氢酶活性影响如图10所示,图中对照组OD值为3.12,MBC组OD值为1.08(仅为对照组的1/3),呼吸链脱氢酶酶活力随提取物浓度的升高而降低,说明经狭果茶藨子提取物处理后蜡样芽孢杆菌中呼吸链脱氢酶活性显著下降,提取物可能通过抑制蜡样芽孢杆菌呼吸链脱氢酶活力,进而抑制呼吸作用和能量供应,最终导致菌体细胞死亡。
2.10扫描电镜观察结果
不同浓度提取物对蜡样芽孢杆菌微观形态影响如图11所示,对照组蜡样芽孢杆菌呈杆状,无异常凸起或凹陷,细胞形态完整,表面光滑,MBC组细胞褶皱,表面有严重凸起和凹陷,细胞严重变形,MIC组细胞呈杆状,表面有褶皱,表面不光滑,1/2MIC组部分细胞有褶皱、但形态完整,变形程度MBC组>MIC组>1/2MIC组>对照组,即变形程度与提取物浓度呈正相关,提取物浓度越高,细胞变形越明显。
3结论
本研究以青藏高原狭果茶藨子为原料,探讨其对蜡样芽孢杆菌的抑菌活性及作用机制,研究表明,狭果茶藨子提取物对蜡样芽孢杆菌有明显抑制作用,乙酸乙酯萃取物为抑制蜡样芽孢杆菌的有效极性部位,且有效活性物质主要存在于DW1组分中,当其浓度为300mg/mL时,抑菌圈直径达18.54±0.52mm,表现为极敏;狭果茶藨子提取物对蜡样芽孢杆菌的最低抑菌浓度和最低杀菌浓度分别为3.13mg/mL和6.25mg/mL。初步抑菌机制试验表明,狭果茶藨子提取物可以改变蜡样芽孢杆菌细胞膜膜蛋白的构象,造成核酸和蛋白质泄露,破坏细胞壁,导致碱性磷酸酶流出,通过抑制琥珀酸脱氢酶、苹果酸脱氢酶、呼吸链脱氢酶的活力抑制呼吸作用和能量代谢,同时提高Na+/K+-ATP酶、Ca2+/Mg2+-ATP酶活力,激活膜结合离子通道,细胞膜内外离子浓度被改变以抵抗不利环境的影响;扫描电镜结果表明狭果茶藨子提取物可导致蜡样芽孢杆菌细胞严重变形,表面有异常凸起和凹陷,最终导致菌体细胞死亡。
实施例7狭果茶藨子提取物在生猪肉保鲜中的应用
7.1将新鲜猪肉切为50g左右的小块,配制狭果茶藨子提取物DW1溶液,使其浓度分别为4MIC(25mg/mL)、2MIC(12.5mg/mL)、MIC(6.25mg/mL)、1/2MIC(3.13mg/mL),取适量均匀涂抹于肉块表面,对照组涂抹灭菌蒸馏水,存放于4℃冰箱,在0、3、6、9、12天时各取1g测量菌落总数,并在第12天对肉块色泽、气味和组织状态进行感官检验,评分标准如表2所示。
表2生猪肉感官评分标准
7.2狭果茶藨子提取物DW1对生猪肉菌落总数的影响
狭果茶藨子提取物DW1对生猪肉菌落总数的影响如图12所示,由图可知,所有实验组中生猪肉菌落总数均随时间延长而增长,但未涂抹狭果茶藨子提取物的空白对照组增长最快,第12天时,空白对照组菌落总数为3945,4MIC组(浓度25mg/mL)菌落总数为2346,比对照组的菌落总数降低了40.5%,与对照组第9天的菌落总数相当,由此说明狭果茶藨子提取物对生猪肉菌落生长有一定抑制作用,可以延缓生猪肉的腐败变质,延长货架期。
7.3狭果茶藨子提取物DW1对生猪肉高铁肌红蛋白比例的影响
狭果茶藨子提取物对生猪肉高铁肌红蛋白比例的影响如图13所示,由图可知,所有实验组生猪肉的高铁肌红蛋白比例均随时间增加而增加,空白对照组增加更为明显,第12天时,其高铁肌红蛋白比例为68.34%,而4MIC组高铁肌红蛋白比例为48.22%,比空白对照组降低了20.12%。高铁肌红蛋白比例越高,肉色越暗,由此说明狭果茶藨子提取物DW1具有保护肉色的作用,可以延缓生猪肉腐败变质,延长货架期。
7.4狭果茶藨子提取物DW1对生猪肉感官评分的影响
狭果茶藨子提取物DW1对生猪肉感官评分的影响如表3所示,由表可知,提取物浓度与感官评分呈正相关,空白对照组色泽评分为9.92分,MIC组评分为15.93分,4MIC组评分为20.34分(是对照组的2.05倍);空白对照组气味评分为10.73,MIC组评分为15.71分,4MIC组评分为22.34分,为对照组的2.08倍;空白对照组组织状态评分为20.14,MIC组评分为28.81分,4MIC组评分为34.79,为对照组1.73倍,由此说明,狭果茶藨子提取物对生猪肉的色泽、气味、组织状态具有良好的保护作用,保鲜效果良好,可在一定程度上延缓生猪肉的腐败变质,延长货架期。
表3狭果茶藨子提取物DW1对生猪肉感官评分的影响
7.5小结
生猪肉保鲜试验发现,狭果茶藨子提取物DW1能抑制生猪肉中腐败菌的生长,延缓其腐败变质,保护肉色稳定,延长货架期,保鲜效果良好。
Claims (10)
1.一种茶藨子提取物,其特征在于:它由包括如下内容的方法制备得到:
取茶藨子乙酸乙酯提取物,上苯乙烯型弱极性大孔吸附树脂,以乙醇含量0-20%的水洗脱,收集洗脱液,制备得到茶藨子提取物。
2.根据权利要求1所述的茶藨子提取物,其特征在于:所述茶藨子选自狭果茶藨子。
3.根据权利要求1所述的茶藨子提取物,其特征在于:所述苯乙烯型弱极性大孔吸附树脂选自AB-8型大孔吸附树脂。
4.根据权利要求1所述的茶藨子提取物,其特征在于:所述乙醇含量0-20%的水中,乙醇含量选自0、10%或20%。
5.根据权利要求1所述的茶藨子提取物,其特征在于:所述洗脱,选自单次洗脱或梯度洗脱。
6.根据权利要求5所述的茶藨子提取物,其特征在于:所述梯度洗脱,指先用乙醇含量为0的水进行洗脱,再使用乙醇含量为20%的水洗脱。
7.茶藨子提取物的制备方法,其特征在于:它包括如下内容:
取茶藨子乙酸乙酯提取物,上苯乙烯型弱极性大孔吸附树脂,以乙醇含量0-20%的水洗脱,收集洗脱液,制备得到茶藨子提取物。
8.权利要求1-6任意一项所述茶藨子提取物在制备抗细菌产品中的用途;进一步地,所述细菌选自蜡样芽孢杆菌。
9.一种抗菌产品,其特征在于:它包括权利要求1-6任意一项所述茶藨子提取物。
10.一种防腐剂,其特征在于:它包括权利要求1-6任意一项所述茶藨子提取物。
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Publication number | Priority date | Publication date | Assignee | Title |
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CN114082222A (zh) * | 2021-10-25 | 2022-02-25 | 青海大学 | 狭果茶藨子游离氨基酸纯化方法 |
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